Hello,
we are conducting cytotoxicity tests since over an year and aren't able to get a stable test. We are no in the validation phase but there are some effects we can't explain. Here is an overview how we conduct the test in compliance with DIN EN ISO 10993-5:
- Seeding 1 x 10^4 cells in a 96-well plate (except the outer wells) --> incubate for 24 h
- Adding medical device extracts (100%, 50%, 10%, 1%) and controls:
- positive control: 1% Triton X (P)
- negative control: cell media (N)
- blank (B): media that was shaken with cell culture media for 24 h at 37 °C in the same kind of container like the medical device was shaken (in compliance with DIN EN ISO 10993-12)
- Incubation for 24 h
- Discard the media and add 50 µl MTT solution (10 mg/ml) --> incubate for 2 h
- Discard and add 100 µl Isopropanol --> incubate for 30 min
- Read out with microplate reader
See the pictures below...the cells are all the time okay until extracts and controlls are added...So something must happen at this point. Sometimes the negative control dies randomly. Sometimes the blank dies. Sometimes only one extract dilution which makes no sense. Does anyone have an idea?
Many thanks!
I have had enough failures in doing cytotoxicity and viability assays to be able to share some insights.
1. I don't know your cell line but if the cells are average sized and multiply properly, I would start by seeding a lower cell density around 1-2k cells per well. Lower cells density allows us to see a better resolution of effect by the agent/ controls on cell survival/viability/proliferation.
2. The most common but less discussed issue is changing media. Everyone you change media due to pipetting a large chunk of cells may be pipetted out. Resulting in negative results in one of the agents or positive controls. Many a times when cell density is very high a large chunk of cells gets uprooted out easily. So without touching the bottom the pipette needs to be touching the edge to gently remove media. This should be practiced throughout all media changes.
3. Another alternative is to avoid change of media as much as possible. One way is to directly put the MTT in the media. Although media has a colour due to phenol red it may affect reading but the media is present in all wells so it should not be an issue. Although some people use a media without phenol red to avoid this issue.
Hope this helps.
I suggest you to check the normal growth of your cells at different time points from different passages. Since your negative controls cells are also dying, I suspect the cells used might be a problem. Cells proliferate at different passages and also at different seeding density due to contact inhibition. I have not tried the same reagent as yours but I have used cell counting kit-8 from Dojindo and got reproducible results everytime.
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